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Contents 1 Protocol 1.1 Medium for drosophila 1.2 M9 medium 1.3 LB medium 1.4 SOB medium 1.5 SOC medium 1.6 Buffer TB 1.7 DNS reaegnt 1.7.1 PCR 1.7.1.1 Standard PCR 1.7.1.2 Screening PCR 1.7.1.3 Mutagenesis (Point mutation, Deletion, Insertion) 1.7.1.4 PCR Purification

Protocol

Medium for drosophila

[http://www.biol.se.tmu.ac.jp/fly/www/standard-medium.html Medium for drosophila]

Materials	Methods
water : 500 mL dry yeast : 20 g corn flour : 45 g glucose : 50 g agarose : 3.5~5 g propionic acid : 1.5 mL 10 % p-hydroxybenzoate in 70 % Eternol : 5 g	Stir dry yeast and agarose with about two-thirds of water. Then, autoclave it. Stir corn flour and glucose with the remaining water. Stir 1 and 2, then autoclave it again. after autoclave, add propionic acid and 10 % p-hydroxybenzoate in 70 % Eternol into it.■

M9 medium

Materials	Methods
water : 1 L Na2HPO4: 6 g KH2PO4 : 3 g NaCl : 0.5 g NH4Cl : 1 g 100 mM MgSO4 : 10 ml 20 % glucose : 10 mL 10 mM CaCl2 : 10 ml 100 mM thiamine-HCl : 10 ml 20 % casamino acid : 10 ml	Stir Na2HPO4, KH2PO4, NaCl and NH4Cl with water. After autoclave, add 10 ml filter sterilized 100 mM MgSO4, 20 % glucose, 10 mM CaCl2, 100 mM thiamine-HCl. If you need, add 10 ml filter sterilized 20 % casamino acid.

LB medium

Materials	Methods
water : 1 L Tryptone: 10 g Yeast extract : 5 g NaCl : 10 g Agar : 15g	Stir Tryptone, Yeast extract and NaCl with water. If you make LB plates, add agar. Autoclave.

SOB medium

Materials	Methods
water : 100 mL Tryptone: 2 g Yeast extract : 0.5 g 5 M NaCl : 200 ul 3 M KCl : 84 ul 1 M MgSO4 : 1 ml 1 M MgCl2 : 1 ml	Stir Tryptone and Yeast extract with water. Add 200 ul 5 M NaCl and 84 ul 3 M KCl. After autoclave, add 1 ml filter sterilized 10 mM MgSO4 and 10 mM MgCl2.

SOC medium

Materials	Methods
SOB : 100 ml 2 M glucose: 1 ml	Add 2 M glucose to 100 ml SOB.

Buffer TB

Materials	Methods
water : 200 ml PIPES : 3 g CaCl2・2H2O : 0.22 g 3 M KCl : 8.315 ml KOH MnCl2・4H2O : 1.09 g	Stir PIPES and CaCl2・2H2O with 100 ml water. Add 8.315 ml 3 M KCl. Add KOH and adjust pH 6.8. Add MnCl2・4H2O. Add water up to 200 ml. Filter sterilize

DNS reaegnt

Materials	Methods
4.5% NaOH : 30 ml 1% DNS : 88 ml Rochelle salt : 25.5 g 10% NaOH : 2.2 ml Phenol : 1 g Water : 7.8 ml NaHCO3 : 0.69 g	Add 1% DNS 88ml and Rochelle salt 25.5 g to 4.5% NaOH 30 ml (A solution) Add Phenol 1 g and water 7.8 ml to 10% NaOH 2.2 ml (B solution) Add NaHCO3 6.9g to B solution 6.9 ml Add A solution 118ml to B solution 6.9 ml Leave 2 days Store in brown bottle

<a name="PCR"></a>

PCR

<a name="Standard_PCR"></a>

Standard PCR

1. Dilute template DNA. If the concentration of DNA is 2-100ng/µL, transfer 1µL to a clean tube and add 99µL MilliQ.
2. Dilute Primer. If the concentration of Primer is XµM, dilute primer X timers and transfer 1µL to a clean tube and add 99µL MilliQ.
3. Mix the following.
   1. For use of KOD plus ver2:

      25mM MgSO4</dt>

      3µL </dd>

      2mM dNTPs</dt>

      5µL </dd>

      10xBuffer for KOD plus ver.2</dt>

      5µL </dd>

      Template DNA (5ng/µL)</dt>

      5µL </dd>

      Primer Forward (10µM)</dt>

      1.5µL </dd>

      Primer Reverse (10µM)</dt>

      1.5µL </dd>

      KOD plus ver.2</dt>

      1µL </dd>

      MilliQ</dt>

      28µL </dd>

      Total</dt>

      50µL </dd>

   2. For use of KOD FX:

      2mM dNTPs</dt>

      10µL </dd>

      2xBuffer for KOD FX</dt>

      25µL </dd>

      Template DNA</dt>

      5µL </dd>

      Primer Forward (10µM)</dt>

      1.5µL </dd>

      Primer Reverse (10µM)</dt>

      1.5µL </dd>

      KOD FX</dt>

      1µL </dd>

      MilliQ</dt>

      6µL </dd>

      Total</dt>

      50µL </dd>

4. (If amplification is not succeeded, try at 4.5 or 6.0µL 25mM MgSO4.)
5. Let stand for 2min at 94℃.
6. 25-40 cycles for 10s at 98℃, for 30s at Tm-5℃, and for 1min (1min for 1kb) at 68℃ (Tm is temparature at which primer will dissolve).
7. At 15℃ forever.
8. Agarose Gel Electrophoresis for confirmation.

<a name="Screening_PCR"></a>

Screening PCR

1. Mix the following (Do on PCR Bench).

   10x PCR buffer (TAKARA)</dt>

   40µL </dd>

   2.5mM dNTP</dt>

   8µL </dd>

   Primer-1 (10pmol/µL)</dt>

   8µL </dd>

   Primer-2 (10pmol/µL)</dt>

   8µL </dd>

   Ex Taq HS (TAKARA)</dt>

   1.6µL </dd>

   MilliQ </dt>

   334µL (to total 400µL) </dd>

2. Dispense 25µL to 15 tubes.
3. Pick a single colony and transfer it to each tubes.
4. Suspend the colony.
5. Let stand for 10min at 90℃.
6. 35 cycles for 30s at 94℃, for 30s 55℃, and for 1min at 72℃.
7. Let stand for 4min at 72℃.
8. Add 5mL Loading Buffer to the tubes.
9. Agalose Gel Electrophoresis for confirmation.
10. Negative Control: Use nothing.
11. Positive Control: Use a colony that will yield a product with this primers.

<a name="Mutagenesis_.28Point_mutation.2C_Deletion.2C_Insertion.29"></a>

Mutagenesis (Point mutation, Deletion, Insertion)

1. Mix the following.

   10xBuffer</dt>

   5µL </dd>

   2mM dNTP</dt>

   5µL </dd>

   Primer Forward (10µM)</dt>

   1.5µL </dd>

   Primer Reverse (10µM)</dt>

   1.5µL </dd>

   Template Plasmid (50ng/µL)</dt>

   1µL </dd>

   KOD plus ver.2</dt>

   1µL </dd>

   MilliQ</dt>

   35µL </dd>

   Total</dt>

   50µL </dd>

2. Prepare control: instead of KOD plus ver.2, add 1µL MilliQ.
3. Let stand for 2min at 94℃.
4. X cycles (1 cycle for 1kb) for 10s at 98℃ and for Ymin (1min for 1kb) at 68℃.
5. Hold at 4℃.
6. Take 25µL of the solutions into fresh tubes.
7. Add 1µL DpnI (10U/µL).
8. Let stand for 1h at 37℃.
9. Agarose gel electrophoresis, using 5µL of the solution for confirmation.
10. Mix the following.

    Sample</dt>

    2µL </dd>

    Ligation high</dt>

    5µL </dd>

    T4 Polynucleotide Kinase (5U/µL)</dt>

    1µL </dd>

    MilliQ</dt>

    7µL </dd>

    Total</dt>

    15µL </dd>

11. Let stand for 1h at 16℃.
12. Transformation, using 10µL of the solution.

<a name="PCR_Purification"></a>

PCR Purification

1. Use QIAquick PCR Purification Kit Cat. No. 28104 by QIAGEN
2. Add BufferPB about 5 times as much as the product of PCR.
3. Apply the solution to the column.
4. Centrifuge for 1min at 1300rpm. Discard the flow-through. If the amount of the sample is much, repeat this step by same column.
5. Add 750µL BufferPE and let stand for 2-3min. If the solution overflows, we decrease the amount of BufferPE.
6. Centrifuge for 1min and discard the through.
7. Centrifuge for additional 1min to remove residual buffer.
8. Place the column in a clean tube.
9. Add 10µL BufferEB or MilliQ to the center of each column, let stand for 1min, and repeat this step.
10. Centrifuge for 1min at 13000rpm.
11. Discard the column.